1 Nature Reviews Microbiology 2005 Vol: 3(8):632-642. DOI: 10.1038/nrmicro1206

Hypovirulence: Mycoviruses at the fungal–plant interface

Whereas most mycoviruses lead 'secret lives', some reduce the ability of their fungal hosts to cause disease in plants. This property, known as hypovirulence, has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. Using one pathogen to control another is appealing, both intellectually and ecologically. The recent development of an infectious cDNA-based reverse genetics system for members of the Hypoviridae mycovirus family has enabled the analysis of basic aspects of this fascinating virus–fungus–plant interaction, including virus–host interactions, the mechanisms underlying fungal pathogenesis, fungal signalling pathways and the evolution of RNA silencing. Such systems also provide a means for engineering mycoviruses for enhanced biocontrol potential.

Mentions
Figures
Figure 1: Taxonomic families and primary modes of hypovirulence-associated mycovirus transmission.a | Taxonomic families represented by mycoviruses that are associated with hypovirulence of plant pathogenic fungi (see Table 1) are shown with the virus structure and genome composition. Totiviruses and chrysoviruses form isometric particles 30–35 nm in diameter, whereas the double-shelled fungal reovirus particles are 80 nm in diameter. b | Mycoviruses are not infectious by an extracellular route. Transmission is restricted primarily to intracellular routes that include cytoplasmic exchange during anastomosis (fusion of hyphae) or during the formation of spores. However, the Cryphonectria parasitica mitovirus NB631 has been shown to be efficiently transmitted to sexual spores (ascospores)117. dsRNA, double-stranded RNA; ssRNA, single-stranded RNA. Figure 2: Genetic organization and basic expression strategy for prototypic hypovirus CHV1-EP713.The coding strand RNA of hypovirus CHV1-EP713 consists of 12,712 nucleotides (nts), excluding the poly(A) tail15. The 5' proximal coding domain, ORF A (composed of 622 codons), encodes two polypeptides, p29 and p40 that are released from a polyprotein, p69, by an autoproteolytic event mediated by p29 (arrowed). During translation, cleavage occurs between Gly248 and Gly249 of the target sequence RIG GRL, and is dependent on Cys162 and His215 in the putative p29 catalytic site58, 59. Expression of ORF B (composed of 3,165 codons) also involves an autoproteolytic event (arrowed) in which a 48-kD polypeptide, p48, is released from the N-terminal portion of the encoded polyprotein. Cleavage, in this case, occurs between Gly418 and Ala419 of the target sequence LVG AEE (Ref. 118). The junction between ORF A and ORF B consists of the sequence 5'-UAAUG-3'. Translational mapping studies indicated that the UAA portion of the pentanucleotide serves as a termination codon of ORF A, whereas the AUG portion is the 5' proximal initiation codon for ORF B, as indicated by the overlapping UAA/AUG codons. Computer-assisted analysis revealed five distinct domains in the CHV1-EP713 coding regions that showed significant sequence similarity with previously described domains, including polymerase (pol) and helicase (hel) domains, in plant potyvirus-encoded polyproteins27. Modified with permission from Ref. 75 © (2001) Annual Reviews. Figure 3: Hypovirus reverse genetics.The figure illustrates the basic features of the Cryphonectria parasitica transformation (left) and transfection (right) protocols. For both protocols, cell-wall-free spheroplasts are produced from virus-free C. parasitica strains. For transformation, a plasmid that contains a full-length hypovirus cDNA copy, such as pXH9, is introduced into spheroplasts by DNA-mediated transformation. The viral cDNA in the transformation vector is fused downstream of the C. parasitica glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and upstream of the GPD terminator. The transformation plasmid also contains the Escherichia coli hygromycin B phosphotransferase gene as a selectable marker flanked by the Aspergillus nidulans trpC promoter and terminator domains. Transformants that contain chromosomally-integrated pXH9 DNA are selected following cell-wall regeneration and growth in the presence of 40 g ml-1 hygromycin B. Transformants contain chromosomally-integrated pXH9 and cDNA-derived cytoplasmically replicating double-stranded (ds)RNA. The hypovirus transfection system uses synthetic transcripts that correspond to the 12.7-kb coding strand of the prot pic hypovirus CHV1-EP713 that are synthesized in a T7-polymerase-dependent cell-free transcription reaction. The synthetic transcripts are introduced into spheroplasts by electroporation and are regenerated in a Petri dish surrounded by molten regeneration agar. As the hyphal structures that are regenerated from the transfected spheroplasts fuse at a high frequency, the resulting dense colony is a large cytoplasmic network. Consequently, replicating hypovirus RNA can migrate throughout the colony. Infected mycelia (transfected hypovirulent strain) are easily obtained by transferring a portion of the colony to a new Petri dish. Modified with permission from Ref. 119 © (2002) CRC. Figure 4: Effect of hypovirus infection on Cryphonectria parasitica colony and canker morphologies.a | Virus-free strain EP155 is shown in the left panel. Isogenic strains that are infected with severe hypovirus CHV1-EP713 and mild hypovirus CHV1-Euro7 are shown in the middle and right panels, respectively. The colonies were grown for 5 days on potato dextrose agar at a temperature of 22°C to 25°C and a light intensity of approximately 2,000 Lux. b | Comparison of canker morphologies formed by hypovirus-infected C. parasitica. A typical canker caused by virus-free strain EP155 is shown in the left panel, whereas cankers caused by strain EP155 after infection by severe hypovirus CHV1-EP713 and by mild hypovirus CHV1-Euro7 are shown in the middle and right panels, respectively. Orange pigmented, spore-producing stromal pustules are prominent features on the surface of cankers formed by virus-free strain EP155 and the isogenic CHV1-Euro7-infected strain, but are rarely formed on the small cankers incited by CHV1-EP173-infected strains. The ridged margins that are characteristic of cankers formed by CHV1-Euro7-infected strains are indicative of callus formation. Cankers were photographed 30 days post-inoculation after wetting to enhance colour contrast of the cankers and surrounding area. Modified with permission from Ref. 75 © (2001) Annual Reviews. Figure 5: Hypovirus symptom determinants and modulation of fungal–plant interactions.a | Emerging map of CHV1-EP713 symptom-determinant domains and essential or dispensable replication elements as described in the text. Reproduced with permission from Ref. 75 © (2001) Annual Reviews. b | Chimeric virus R12 contains the region of ORF B that extends from the N-terminus of p48 (nucleotide (nt) 2,363) to the Nar I site at nt 5,310 from the severe hypovirus CHV1-EP713 and the remainder of ORF B from the mild hypovirus CHV1-Euro7. Chimeric virus R6 contains the reciprocal arrangement. c | Cankers caused by Cryphonectria parasitica strain EP155 infected with chimeric CHV1-EP713/CHV1-Euro7 viruses R12 and R6. The canker caused by R12-infected strain EP155 resembles cankers formed by CHV1-EP713-infected strains whereas the canker formed by R6-infected strain EP155 is small, but has a surface and margins similar to cankers formed by CHV1-Euro7-infected strains. Modified with permission from Ref. 45 © (2000) American Society for Microbiology.
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References
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    • . . . Mycoviruses are widespread throughout the major taxonomic groups of fungi, and persistently infect their hosts, usually without any discernable phenotypic changes1 . . .
    • . . . Mycoviruses are not infectious in the classic sense1, as infection cannot be initiated by exposing uninfected hyphae to a cell extract that has been prepared from an infected strain . . .
  2. Anagnostakis, S. L. Biological control of chestnut blight. Science 215, 466-471.One of the first reviews of the use of hypovirulence as a biological control strategy and one of the best descriptions of the North American chestnut blight epidemic , (1982) .
    • . . . The introduction of the chestnut blight fungus Cryphonectria parasitica into North America and Europe from Asia resulted in the destruction of billions of mature American and European chestnut trees during the first half of the last century2 . . .
    • . . . Efforts to establish effective biocontrol in North American forest ecosystems by the artificial introduction of hypovirulent C. parasitica strains have so far been unsuccessful2, 95, 96 . . .
    • . . . This provides an unparalleled therapeutic treatment of individual cankers and is one of the few methods that prevents chestnut blight from killing individual infected trees2 . . .
    • . . . Hypovirus RNA is not transmitted to ascospores during mating2 . . .
  3. Brasier, C. M., in The Elms: Breeding, Conservation and Disease Management (ed. Dunn, C. P.) 61-72 (Kluwer, Boston, 2000).Excellent source for information on Dutch elm disease and the hypovirulence-associated viruses of O. ulmi and O. novo-ulmi , .
    • . . . The ravages of the Dutch elm disease fungi Ophiostoma ulmi and Ophiostoma novo-ulmi have changed the landscape of our city streets3 . . .
  4. Ghabrial, S. A., Soldevila, A. I. & Havens, W. M. in Molecular Biology of double-stranded RNA: Concepts and Applications in Agriculture, Forestry and Medicine (ed. Tavantzis, S.) 213-236 (CRC, Boca Raton, 2002) , .
    • . . . All hypovirulence-associated mycoviruses have double-stranded (ds) or single-stranded (ss)RNA genomes and include representatives of the Totiviridae and Chrysoviridae4 (common mycovirus families), and two members of the Reoviridae5, 6 . . .
    • . . . Interestingly, hypovirulent strains of the oat pathogen Helminthosporium victoriae harbour both a totivirus (Hv190SV) and a chrysovirus (Hv145SV)4 . . .
  5. Wei, C. Z., Osaki, H., Iwanami, T., Matsumoto, N. & Ohtsu, Y. Molecular characterization of dsRNA segments 2 and 5 and electron microscopy of a novel reovirus from a hypovirulent isolate, W370, of the plant pathogen Rosellina necatrix. J. Gen. Virol. 84, 2431-2437 , (2003) .
    • . . . All hypovirulence-associated mycoviruses have double-stranded (ds) or single-stranded (ss)RNA genomes and include representatives of the Totiviridae and Chrysoviridae4 (common mycovirus families), and two members of the Reoviridae5, 6 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  6. Suzuki, N., Supyani, S., Maruyama, K. & Hillman, B. I. Complete genome sequence of Mycoreovirus-1/Cp9B21, a member of a novel genus within the family Reoviridae, isolated from the chestnut blight fungus Cryphonectria parasitica. J. Gen. Virol. 85, 3437-3448 , (2004) .
    • . . . All hypovirulence-associated mycoviruses have double-stranded (ds) or single-stranded (ss)RNA genomes and include representatives of the Totiviridae and Chrysoviridae4 (common mycovirus families), and two members of the Reoviridae5, 6 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
    • . . . Exceptions include the C. parasitica reovirus Cp9B21 (Ref. 6) and an unclassified virus from Botrytis cinerea25 — here, infections were established by introducing purified viral particles into fungal spheroplasts . . .
  7. Wickner, R. B., Wang, C. C. & Patterson, J. L. in Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses (eds Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A.) 571-580 (Elsevier, London, 2005) , .
    • . . . Totiviruses and chrysoviruses form isometric particles that contain monopartite or tetrapartite dsRNA genomes, respectively7, 8 whereas the fungal reoviruses contain 11 individual dsRNA segments packaged in double-shelled particles that are 80 nm in diameter9. . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  8. Ghabrial, S. A., Jiang, D. & Caston, J. R. in Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses (eds Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A.) 591-595 (Elsevier, London, 2005) , .
    • . . . Totiviruses and chrysoviruses form isometric particles that contain monopartite or tetrapartite dsRNA genomes, respectively7, 8 whereas the fungal reoviruses contain 11 individual dsRNA segments packaged in double-shelled particles that are 80 nm in diameter9. . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  9. Mertens, P. P. C., Hillman, B. I., & Suzuki, N. in Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses (eds Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A.) (in the press) (Elsevier, London, 2005) , .
    • . . . Totiviruses and chrysoviruses form isometric particles that contain monopartite or tetrapartite dsRNA genomes, respectively7, 8 whereas the fungal reoviruses contain 11 individual dsRNA segments packaged in double-shelled particles that are 80 nm in diameter9. . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  10. Wickner, R. B., Esteban, R. & Hillman, B. I. in Virus Taxonomy: VIIth report of the International Committee on Taxonomy of Viruses. (eds Van Regenmortel, M. H. et al.) 651-656 (Academic, San Diego, 2000) , .
    • . . . Members of a new family of naked ssRNA viruses, the Narnaviridae10, in the genus Mitovirus, have been isolated from three different plant-pathogenic fungi, C. parasitica11, O. ulmi12 and Sclerotinia homoeocarpa13 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  11. Polashock, J. J. & Hillman, B. I. A small mitochondrial double-stranded (ds) RNA element associated with a hypovirulent strain of the chestnut blight fungus and ancestrally related to yeast cytoplasmic T and W dsRNAs. Proc. Natl Acad. Sci. USA 91, 566-571 , (1994) .
    • . . . Members of a new family of naked ssRNA viruses, the Narnaviridae10, in the genus Mitovirus, have been isolated from three different plant-pathogenic fungi, C. parasitica11, O. ulmi12 and Sclerotinia homoeocarpa13 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  12. Hong, Y., Dover, S. L., Cole, T. E., Brasier, C. M. & Buck, K. W. Multiple mitochondrial viruses in an isolate of the Dutch elm disease fungus Ophiostoma novo-ulmi. Virology 246, 158-169 , (1999) .
    • . . . Members of a new family of naked ssRNA viruses, the Narnaviridae10, in the genus Mitovirus, have been isolated from three different plant-pathogenic fungi, C. parasitica11, O. ulmi12 and Sclerotinia homoeocarpa13 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  13. Deng, F., Xu, R. & Boland, G. J. Hypovirulence-associated double-stranded RNA from Sclerotinia homoeocarpa is conspecific with Ophiostoma novo-ulmi mitovirus 3a-Ld. Phytopathology 93, 1407-1414 , (2003) .
    • . . . Members of a new family of naked ssRNA viruses, the Narnaviridae10, in the genus Mitovirus, have been isolated from three different plant-pathogenic fungi, C. parasitica11, O. ulmi12 and Sclerotinia homoeocarpa13 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  14. Cole, T. E., Hong, Y., Brasier, C. M. & Buck, K. W. Detection of an RNA-dependent RNA polymerase in mitochondria from a mitovirus-infected isolate of the Dutch Elm Disease fungus Ophiostoma novo-ulmi. Virology 268, 239-243 , (2000) .
    • . . . These small (<3kb) viral RNAs seem to replicate in association with the mitochondria and encode a single polypeptide that serves as an RNA-dependent RNA polymerase (RDRP)14 . . .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  15. Shapira, R., Choi, G. H. & Nuss, D. L. Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight. EMBO J. 10, 731-739 , (1991) .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
    • . . . Although hypovirus RNA is found in hyphal extracts as dsRNA, the structural characteristics of the dsRNA are reminiscent of a replicative intermediate or replicative form of a ssRNA virus15 . . .
    • . . . The coding strand RNA of hypovirus CHV1-EP713 consists of 12,712 nucleotides (nts), excluding the poly(A) tail15 . . .
    • . . . The complete nucleotide sequence of CHV1-EP713 (Ref. 15) and the timely report of a robust DNA-mediated genetic transformation protocol for C. parasitica33 facilitated this approach . . .
  16. Hillman, B. I., Tian, Y., Bedker, P. J. & Brown, M. P. A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA. J. Gen. Virol. 73, 681-686 , (1992) .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  17. Chen, B. & Nuss, D. L. Infectious cDNA clone of hypovirus CHV1/Euro7: a comparative virology approach to investigate virus-mediated hypovirulence of the chestnut blight fungus Cryphonectria parasitica. J. Virol. 73, 985-992 , (1999) .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
    • . . . Both viruses also reduce the ability of cultured mycelia to produce CONIDIA, but the reduction in sporulation is more extensive for CHV1-EP713 than for CHV1-Euro7 (Ref. 17) . . .
    • . . . However, C. parasitica cultures that are infected with CHV1-Euro7 have a faster growth rate than isogenic, virus-free cultures, whereas cultures infected with CHV1-EP713 have a slower growth rate and form irregular colony margins when grown on solid medium17, 45. . . .
    • . . . The resulting cankers are covered with stromal pustules that contain viable asexual spores17 . . .
    • . . . Importantly, the independent introduction of infectious transcripts of each hypovirus into several C. parasitica strains showed that the viral genomes predominantly contribute to the expression of symptoms, with minor, but measurable, contributions from the host genome17. . . .
    • . . . Initial studies mapped the differences in symptom expression to ORF B and indicated that ORF A makes similar contributions to the overall level of symptom expression17 . . .
    • . . . The availability of an infectious cDNA clone for the mild hypovirus CHV1-Euro7 (Ref. 17) now provides the means to construct a transgenic hypovirulent strain that combines the properties of enhanced colonization and spore production with a novel mode of hypovirus transmission to ascospore progeny . . .
  18. Smart, C. D. et al. Cryphonectria hypovirus 3, a virus in the family Hypoviridae with a single open reading frame. Virology 265, 66-73 , (1999) .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  19. Linder-Basso, D., Dynek, J. & Hillman, B. I. Genome analysis of Cryphonectria hypovirus 4, the most common hypovirus species in North America. Virology (in the press) , .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  20. Preisig, O., Moleleki, N., Smit, W. A., Wingfield, B. D. & Wingfield, M. J. A novel RNA mycovirus in a hypovirulent isolate of the plant pathogen Diaporthe ambigua. J. Gen. Virol. 81, 3107-3114 , (2000) .
    • . . . Members of the Hypoviridae family5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and an unclassified viral RNA that is associated with hypovirulent strains of Diaporthe perjuncta20 not only lack capsid proteins, but are phylogenetically related to the ssRNA plant viruses potyviruses and tombusviruses, respectively . . .
  21. Lakshman, D. K., Jian, J. & Tavantzis, S. M. A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway. Proc. Natl Acad. Sci. USA 95, 6425-6429 , (1998) .
    • . . . Other unclassified viral RNAs that are associated with hypovirulence in Rhizoctonia solani21 and Fusarium graminearum22 also seem to lack capsid proteins . . .
  22. Chu, Y. -M. et al. Double-stranded RNA mycovirus from Fusarium graminearum. Appl. Environ. Microbiol. 68, 2529-2534 , (2002) .
    • . . . Other unclassified viral RNAs that are associated with hypovirulence in Rhizoctonia solani21 and Fusarium graminearum22 also seem to lack capsid proteins . . .
  23. Buck, K. W. in Molecular Variability of Fungal Pathogens (eds Bridge, P. D., Couteaudier, Y. & Clarkson, J. M.) 53-72 (CAB International, Wallingford, 1998).This review provides an excellent overview of the genome organization and expression strategies of fungal viruses , .
    • . . . Rather, mycoviruses are mainly transmitted by cytoplasmic mixing following hyphal fusion (ANASTOMOSIS) or by the formation of asexual spores (Fig. 1; see Refs 23,24 for recent general mycovirus reviews). . . .
    • . . . Disruption of mitochondrial function has been associated with the presence of mitoviruses in slow growing isolates of the Dutch elm disease fungus23 . . .
  24. Ghabrial, S. A. Origin, adaptation and evolutionary pathways of fungal viruses. Virus Genes 16, 119-131.Provides perspectives on the origin and evolution of fungal viruses , (1998) .
    • . . . Rather, mycoviruses are mainly transmitted by cytoplasmic mixing following hyphal fusion (ANASTOMOSIS) or by the formation of asexual spores (Fig. 1; see Refs 23,24 for recent general mycovirus reviews). . . .
  25. Castro, M., Kramer, K., Valdivia, L., Ortiz, S. & Castillo, A. A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea. FEMS Microbiol. Lett. 228, 87-91 , (2003) .
    • . . . Exceptions include the C. parasitica reovirus Cp9B21 (Ref. 6) and an unclassified virus from Botrytis cinerea25 — here, infections were established by introducing purified viral particles into fungal spheroplasts . . .
  26. Hillman, B. I. & Suzuki, N. Viruses of the chestnut blight fungus. Adv. Virus Res. 63, 423-472.A recent and comprehensive review of viruses associated with the chestnut blight fungus C. parasitica , (2004) .
    • . . . The genome organization of these four species has been reviewed26 . . .
    • . . . This correlates with the sequence diversity among CHV species (reviewed in Ref. 26) and might indicate a link between hypovirus genetic diversity and the phenotypic diversity of hypovirulent field isolates . . .
  27. Koonin, E. V., Choi, G. H., Nuss, D. L., Shapira, R. & Carrington, J. C. Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses. Proc. Natl Acad. Sci. USA 88, 10647-10651 , (1991) .
    • . . . Domains in both ORF A and ORF B share sequence homology with ssRNA plant potyviruses27 and the polyadenylated strand is infectious28, which is consistent with a ssRNA viral replication strategy. . . .
    • . . . Computer-assisted analysis revealed five distinct domains in the CHV1-EP713 coding regions that showed significant sequence similarity with previously described domains, including polymerase (pol) and helicase (hel) domains, in plant potyvirus-encoded polyproteins27 . . .
    • . . . These similarities included conserved N-terminal cysteine residues27 and papain-like, protease catalytic and cleavage domains58, 59 . . .
  28. Chen, B., Choi, G. H. & Nuss, D. L. Attenuation of fungal virulence by synthetic infectious hypovirus transcripts. Science 264, 1762-1764 , (1994) .
    • . . . Domains in both ORF A and ORF B share sequence homology with ssRNA plant potyviruses27 and the polyadenylated strand is infectious28, which is consistent with a ssRNA viral replication strategy. . . .
    • . . . RNA transfection (Fig. 3) bypasses the chromosomal integration of hypovirus cDNA and initiates infection by electroporation of a synthetic copy of hypovirus positive strand RNA directly into fungal spheroplasts28 . . .
    • . . . One of the earliest uses of the transfection system was the expansion of the host range of CHV1-EP713 to several fungal species that are closely related to C. parasitica28 . . .
  29. Racaniello, V. R. & Baltimore, D. Cloned poliovirus cDNA is infectious in mammalian cells. Science 214, 916-919 , (1981) .
    • . . . Although infectious cDNAs were constructed from RNA viruses of animals29 and plants30 in the 1980s, it was not until the construction of a full-length infectious cDNA clone of the CHV1-EP713 hypovirus in 1991 that the first reverse genetics system was developed for a mycovirus31 . . .
  30. Ahlquist, P., French, R., Janda, M. & Loesch-Fries, L. S. Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc. Natl Acad. Sci. USA 81, 7066-7070 , (1984) .
    • . . . Although infectious cDNAs were constructed from RNA viruses of animals29 and plants30 in the 1980s, it was not until the construction of a full-length infectious cDNA clone of the CHV1-EP713 hypovirus in 1991 that the first reverse genetics system was developed for a mycovirus31 . . .
  31. Choi, G. H. & Nuss, D. L. Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA. Science 257, 800-803.Describes the construction of an infectious hypovirus cDNA, which established the first mycovirus reverse genetics system , (1992) .
    • . . . Although infectious cDNAs were constructed from RNA viruses of animals29 and plants30 in the 1980s, it was not until the construction of a full-length infectious cDNA clone of the CHV1-EP713 hypovirus in 1991 that the first reverse genetics system was developed for a mycovirus31 . . .
    • . . . In the first strategy, the hypovirus cDNA clone is integrated into the nuclear DNA of C. parasitica, which then gives rise to cDNA-derived, cytoplasmically-replicating viral positive strand RNA31 (Fig. 3) . . .
    • . . . Viral cDNA has been integrated into nuclear DNA by two methods: polyethylene-glycol-mediated uptake by fungal spheroplasts31 and, more recently, by particle bombardment of intact fungal mycelia34 . . .
  32. Moleleki, N., van Heerden, S. W., Wingfield, M. J., Wingfield, B. D. & Preisig, O. Transfection of Diaporthe perjuncta with Diaporthe RNA virus. Appl. Environ. Microbiol. 69, 3952-3956 , (2003) .
    • . . . However, the infectious transcripts that were derived from the cDNA clone failed to confer hypovirulence when used to establish infection in a virus-free strain32 . . .
  33. Churchill, A. C. L., Ciufetti, L. M., Hansen, D. R., Van Etten, H. D. & Van Alfen, N. K. Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Curr. Genet. 17, 25-31 , (1990) .
    • . . . The complete nucleotide sequence of CHV1-EP713 (Ref. 15) and the timely report of a robust DNA-mediated genetic transformation protocol for C. parasitica33 facilitated this approach . . .
  34. Sasaki, A. et al. Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA. Mol. Plant. Microb. Interact. 15, 780-789 , (2002) .
    • . . . Viral cDNA has been integrated into nuclear DNA by two methods: polyethylene-glycol-mediated uptake by fungal spheroplasts31 and, more recently, by particle bombardment of intact fungal mycelia34 . . .
    • . . . More recently, particle bombardment with CHV1-EP713 cDNA was used to introduce this hypovirus into the fruit tree pathogens Phomopsis G-type and Valsa ceratosperma (members of the same taxonomic order as C. parasitica, but classified in separate genera), which resulted in virulence attenuation in both cases34. . . .
  35. Chen, B., Craven, M. G., Choi, G. H. & Nuss, D. L. cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides. Virology 202, 441-448 , (1994) .
    • . . . Chen et al.35 showed that the cytoplasmically-replicating hypovirus RNA that is present in these TRANSGENIC HYPOVIRULENT strains originated as large cDNA-derived nuclear transcripts that were subsequently trimmed to remove nonviral vector nucleotides . . .
  36. van Heerden, S. W. et al. Characterization of South African Cryphonectria cubensis isolates infected with a C. parasitica hypovirus. Phytopathology 91, 628-632 , (2001) .
    • . . . Transfection-mediated introduction of CHV1-EP713 RNA into South African strains of Cryphonectria cubensis, a pathogen of Eucalyptus in plantations, has provided opportunities for biocontrol of Cryphonectria canker disease36 . . .
  37. Elliston, J. E. Characterization of dsRNA-free and dsRNA-containing strains of Endothia parasitica in relation to hypovirulence. Phytopathology 75, 151-158 , (1985) .
    • . . . These other symptoms always include the loss of female fertility in sexual crosses, usually include a reduction in asexual sporulation and altered colony morphologies, and often include modulation of pigment production and the diminished accumulation of specific metabolites such as oxalic acid37, 38, 39 . . .
  38. Anagnostakis, S. L. & Day, P. R. Hypovirulence conversion in Endothia parasitica. Phytopathology 69, 1226-1229 , (1979) .
    • . . . These other symptoms always include the loss of female fertility in sexual crosses, usually include a reduction in asexual sporulation and altered colony morphologies, and often include modulation of pigment production and the diminished accumulation of specific metabolites such as oxalic acid37, 38, 39 . . .
  39. Anagnostakis, S. L. in The Ecology and Physiology of the Fungal mycelium (eds Jennings, D. H. & Rayner, A. D. M.) 353-366 (Cambridge University Press, Cambridge, 1984) , .
    • . . . These other symptoms always include the loss of female fertility in sexual crosses, usually include a reduction in asexual sporulation and altered colony morphologies, and often include modulation of pigment production and the diminished accumulation of specific metabolites such as oxalic acid37, 38, 39 . . .
  40. Peever, T. L., Liu, Y. -C. & Milgroom, M. G. Incidence and diversity of hypoviruses and other double-stranded RNAs occurring in the chestnut blight fungus Cryphonectria parasitica, in China and Japan. Phytopathology 88, 811-17 , (1998) .
    • . . . Surveys of C. parasitica field isolates collected in Asia40, Europe41 and North America42, 43, 44 have revealed considerable variability in the level of hypovirulence and the range of associated symptoms . . .
  41. Allemann, C., Hoegger, P., Heiniger, U. & Rigling, D. Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers. Mol. Ecol. 8, 843-854 , (1999) .
    • . . . Surveys of C. parasitica field isolates collected in Asia40, Europe41 and North America42, 43, 44 have revealed considerable variability in the level of hypovirulence and the range of associated symptoms . . .
    • . . . Consistent with this prediction, a recent survey of hypoviruses in regions of Europe with a high incidence of hypovirulence found little evidence of severe CHV1-EP713-related hypoviruses and a prevalence of mild CHV1-Euro7-related hypoviruses41, 110 . . .
  42. Chung, P., Bedker, P. J. & Hillman, B. I. Diversity of Cryphonectria parasitica hypovirulence-associated double-stranded RNAs within a chestnut population in New Jersey. Phytopathology 84, 984-990 , (1994) .
    • . . . Surveys of C. parasitica field isolates collected in Asia40, Europe41 and North America42, 43, 44 have revealed considerable variability in the level of hypovirulence and the range of associated symptoms . . .
  43. Enebak, S. A., MacDonald, W. L. & Hillman, B. I. Effect of dsRNA associated with isolates of Cryphonectria parasitica from the central Appalachians and their relatedness to other dsRNAs from North America and Europe. Phytopathology 84, 528-534 , (1994) .
    • . . . Surveys of C. parasitica field isolates collected in Asia40, Europe41 and North America42, 43, 44 have revealed considerable variability in the level of hypovirulence and the range of associated symptoms . . .
    • . . . Hypovirus spread is also influenced by the efficiency of transmission through conidia (Fig. 1), which can vary from 1% to 99% depending on the hypovirus–fungal host combination43, 104, 105 . . .
  44. Peever, T. L., Liu, Y. -C. & Milgroom, M. G. Diversity of hypoviruses and other double-stranded RNAs in Cryphonectria parasitica in North America. Phytopathology 87, 1026-1033 , (1997) .
    • . . . Surveys of C. parasitica field isolates collected in Asia40, Europe41 and North America42, 43, 44 have revealed considerable variability in the level of hypovirulence and the range of associated symptoms . . .
  45. Chen, B., Geletka, L. M. & Nuss, D. L. Using chimeric hypoviruses to fine-tune the interaction between a pathogenic fungus and its plant host. J. Virol. 74, 7562-7567 , (2000) .
    • . . . However, C. parasitica cultures that are infected with CHV1-Euro7 have a faster growth rate than isogenic, virus-free cultures, whereas cultures infected with CHV1-EP713 have a slower growth rate and form irregular colony margins when grown on solid medium17, 45. . . .
    • . . . Chen et al.45 subsequently generated a collection of stable ORF B chimeric viruses that caused a spectrum of defined symptoms . . .
    • . . . Modified with permission from Ref. 45 © (2000) American Society for Microbiology. . . .
    • . . . The idea of balancing ecological fitness and virulence attenuation could be further developed through construction of transgenic hypovirulent strains with cDNA clones of CHV1-EP713–CHV1-Euro7 chimaeras45 or new hypovirus isolates as they become available. . . .
  46. Suzuki, N., Geletka, L. M. & Nuss, D. L. Essential and dispensible virus-encoded replication elements revealed by efforts to develop hypoviruses as gene expression vectors. J. Virol. 74, 7568-7577 , (2000) .
    • . . . Efforts to develop hypoviruses as gene expression vectors resulted in the surprising finding that the first 24 codons of ORF A are required for viral replication46 . . .
  47. Craven, M. G., Pawlyk, D. M., Choi, G. H. & Nuss, D. L. Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus. J. Virol. 67, 6513-6521 , (1993) .
    • . . . However, in the absence of viral infection, ectopic expression of the p29 coding domain in the host reduced the level of pigmentation47, 48and conidiation, whereas ectopic expression of p40 had no effect49 . . .
  48. Suzuki, N., Chen, B. & Nuss, D. L. Mapping of a hypovirus p29 protease symptom determinant domain with sequence similarity to potyvirus HC-Pro protease. J. Virol. 73, 9478-9484 , (1999) .
    • . . . However, in the absence of viral infection, ectopic expression of the p29 coding domain in the host reduced the level of pigmentation47, 48and conidiation, whereas ectopic expression of p40 had no effect49 . . .
    • . . . The domain that is responsible for p29-mediated suppression of pigmentation and sporulation was mapped to ORF A (Phe25 to Gln73 (Ref. 48)), whereas the Thr288 to Arg312 domain of ORF A is responsible for p40-mediated accumulation of viral RNA49 . . .
  49. Suzuki, N. & Nuss, D. L. The contribution of protein p40 to hypovirus-mediated modulation of fungal host phenotype and viral RNA accumulation. J. Virol. 76, 7747-7759 , (2002) .
    • . . . Viral RNA accumulation was reduced for both mutants49 . . .
    • . . . However, in the absence of viral infection, ectopic expression of the p29 coding domain in the host reduced the level of pigmentation47, 48and conidiation, whereas ectopic expression of p40 had no effect49 . . .
    • . . . The domain that is responsible for p29-mediated suppression of pigmentation and sporulation was mapped to ORF A (Phe25 to Gln73 (Ref. 48)), whereas the Thr288 to Arg312 domain of ORF A is responsible for p40-mediated accumulation of viral RNA49 . . .
    • . . . Surprisingly, although deletion of all but the first 24 codons of ORF A resulted in a 90% reduction in viral RNA accumulation and a significant relief of virus-mediated suppression of host conidiation, it did not reduce the level of virus-mediated attenuation of fungal virulence49. . . .
  50. Suzuki, N., Maruyama, K., Moriyama, M. & Nuss, D. L. Hypovirus papain-like protease p29 functions in trans to enhance viral double-stranded RNA accumulation and vertical transmission. J. Virol. 77, 11697-11707 , (2003) .
    • . . . A possible role for p29 as a suppressor of RNA silencing has recently been suggested50 . . .
  51. Napoli, C., Lemieux, C. & Jorgensen, R. Introduction of a chalocone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279-289 , (1990) .
    • . . . RNA-mediated, sequence-specific suppression of gene expression, known as RNA silencing, has also been called post-transcriptional gene silencing in plants51, 52, quelling in fungi53 and RNA interference in animals54 . . .
  52. van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. & Stuitje, A. R. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to suppression of gene expression. Plant Cell 2, 291-299 , (1990) .
    • . . . RNA-mediated, sequence-specific suppression of gene expression, known as RNA silencing, has also been called post-transcriptional gene silencing in plants51, 52, quelling in fungi53 and RNA interference in animals54 . . .
  53. Romano, N. & Macino, G. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol. Microbiol. 6, 3343-3345 , (1992) .
    • . . . RNA-mediated, sequence-specific suppression of gene expression, known as RNA silencing, has also been called post-transcriptional gene silencing in plants51, 52, quelling in fungi53 and RNA interference in animals54 . . .
  54. Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-363 , (1999) .
    • . . . RNA-mediated, sequence-specific suppression of gene expression, known as RNA silencing, has also been called post-transcriptional gene silencing in plants51, 52, quelling in fungi53 and RNA interference in animals54 . . .
  55. Voinnet, O. RNA silencing as a plant immune system against viruses. Trends Genet. 17, 449-459 , (2001) .
    • . . . A role for RNA silencing as an antiviral defence mechanism was first shown in plants (reviewed in Ref. 55) and more recently in animal cells56 . . .
  56. Li, H. W., Li, W. X. & Ding, S. W. Induction and suppression of RNA silencing by an animal virus. Science 296, 1319-1321 , (2002) .
    • . . . A role for RNA silencing as an antiviral defence mechanism was first shown in plants (reviewed in Ref. 55) and more recently in animal cells56 . . .
  57. Silhavy, D. & Burgyan, J. Effects and side-effects of viral RNA silencing suppressors on short RNAs. Trends Plant Sci. 9, 76-83 , (2004) .
    • . . . Viruses produce proteins that suppress host cell RNA silencing (reviewed in Ref. 57) . . .
  58. Choi, G. H., Pawlyk, D. M., & Nuss, D. L. The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro. Virology 183, 747-752 , (1991) .
    • . . . During translation, cleavage occurs between Gly248 and Gly249 of the target sequence RIG GRL, and is dependent on Cys162 and His215 in the putative p29 catalytic site58, 59 . . .
    • . . . These similarities included conserved N-terminal cysteine residues27 and papain-like, protease catalytic and cleavage domains58, 59 . . .
  59. Choi, G. H., Shapira, R. & Nuss, D. L. Co-translational autoproteolysis involved in gene expression from a double-stranded RNA genetic element associated with hypovirulence of the chestnut blight fungus. Proc. Natl Acad. Sci. USA 88, 1167-1171 , (1991) .
    • . . . During translation, cleavage occurs between Gly248 and Gly249 of the target sequence RIG GRL, and is dependent on Cys162 and His215 in the putative p29 catalytic site58, 59 . . .
    • . . . These similarities included conserved N-terminal cysteine residues27 and papain-like, protease catalytic and cleavage domains58, 59 . . .
  60. Brigneti, G. O. et al. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17, 6739-6746 , (1998) .
    • . . . Suppression of RNA silencing by p29 has now been demonstrated both in a transgenic Nicotiana benthamiana line60 (Y . . .
  61. Hannon, G. J. RNA interference. Nature 418, 244-251 , (2002) .
    • . . . RNA silencing is part of a larger set of regulatory pathways involving small RNAs that include the microRNAs (miRNAs) involved in developmental regulation in plants and animals61 . . .
  62. Kasschau, K. D. et al. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and mRNA function. Dev. Cell. 4, 205-217 , (2003) .
    • . . . Virus-encoded suppressors of RNA silencing such as HC-Pro62 contribute to virus-induced disease symptoms by interfering with miRNA-controlled developmental pathways . . .
  63. Catalanotto, C. et al. Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol. Cell. Biol. 24, 2536-2545 , (2004) .
    • . . . Essentially, all that is known about RNA silencing in fungi has been learned from the model filamentous fungus Neurospora crassa63, 64, 65 . . .
  64. Chicas, A. & Macino, G. Characteristics of post-transcriptional gene silencing. EMBO Rep. 2, 992-996 , (2001) .
    • . . . Essentially, all that is known about RNA silencing in fungi has been learned from the model filamentous fungus Neurospora crassa63, 64, 65 . . .
  65. Cogoni, C. Homology-dependent gene silencing mechanisms in fungi. Annu. Rev. Microbiol. 55, 381-406.Includes description of the elements of RNA silencing in fungi , (2001) .
    • . . . Essentially, all that is known about RNA silencing in fungi has been learned from the model filamentous fungus Neurospora crassa63, 64, 65 . . .
  66. Dawe, A. L. et al. An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea. Microbiology 149, 2373-2384.Emphasizes the concept of using mycoviruses to understand and manage fungal virulence , (2003) .
    • . . . However, C. parasitica is closely related to N. crassa66 and several C. parasitica homologues of essential components of the N. crassa RNA silencing pathway have been cloned (G . . .
    • . . . As recently as 2002, only 40 C. parasitica genes were deposited in the NCBI (National Center for Biotechnology Information) database, but the construction of an ordered EXPRESSED SEQUENCE TAG (EST) library representing approximately 2,200 C. parasitica genes, that is accessible on the COGEME web site maintained by Exeter University77 (see Online links box), has been a boon to hypovirus research66 . . .
  67. Larson, T. G., Choi, G. H. & Nuss, D. L. Regulatory pathways governing modulation of fungal gene expression by a virulence-attenuating mycovirus. EMBO J. 11, 4539-4548 , (1992) .
    • . . . Larson and co-workers used the laccase gene lac-1 as a reporter to monitor CHV1-EP713-mediated alteration of inositol triphosphate (IP3)/Ca2+/calmodulin signalling67, 68 . . .
  68. Larson, T. G. & Nuss, D. L. Altered transcriptional response to nutrient availability in hypovirus-infected chestnut blight fungus. EMBO J. 13, 5616-5623 , (1994) .
    • . . . Larson and co-workers used the laccase gene lac-1 as a reporter to monitor CHV1-EP713-mediated alteration of inositol triphosphate (IP3)/Ca2+/calmodulin signalling67, 68 . . .
  69. Park, S. M. et al. Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress. Mol. Microbiol. 51, 1267-1277 , (2004) .
    • . . . Recently, Kim and co-workers have provided evidence for the alteration of a C. parasitica mitogen-activated protein kinase (MAPK) cascade that includes CpMK-1 (Ref. 69) and the upregulation of a serine/threonine protein kinase CpPK-1 (Ref. 70) in CHV1-infected C. parasitica . . .
  70. Kim, M. J. et al. Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus. Mol. Microbiol. 45, 933-941 , (2002) .
    • . . . Recently, Kim and co-workers have provided evidence for the alteration of a C. parasitica mitogen-activated protein kinase (MAPK) cascade that includes CpMK-1 (Ref. 69) and the upregulation of a serine/threonine protein kinase CpPK-1 (Ref. 70) in CHV1-infected C. parasitica . . .
  71. Choi, G. H., Chen, B. & Nuss, D. L. Virus-mediated or transgenic suppression of a G-protein subunit and attenuation of fungal virulence. Proc. Natl Acad. Sci. USA 92, 305-309 , (1995) .
    • . . . In one of the first reports to link G-protein signalling and fungal virulence, Choi et al.71 showed that hypovirus infection resulted in the reduced accumulation of a specific G subunit, CPG-1, and that transgenic co-suppression of this subunit correlated with reduced fungal virulence . . .
  72. Dawe, A. L., Segers, G. C., Allen, T. D., McMains, V. C. & Nuss, D. L. Microarray analysis of Cryphonectria parasitica G and G-signalling pathways reveals extensive modulation by hypovirus infection. Microbiology 150, 4033-4043 , (2004) .
    • . . . Reduction in the accumulation of CPG-1 and the cognate G subunit CPGB-1 by CHV1-EP713 was recently confirmed72 . . .
    • . . . Disruption of the gene that encodes CPG-1 resulted in a loss of virulence, reduced accumulation of CPGB-1 and a set of phenotypic changes that are similar to, but more severe than, those caused by hypovirus infection72, 73 . . .
    • . . . Using strains deleted for cpg-1 (G) and cpgb-1(G), more than 250 C. parasitica genes that are potentially regulated by G-protein signalling were identified72 . . .
    • . . . Comparisons of transcriptional profiles revealed that more than half (53.7%) of the CHV1-EP713-responsive genes were also modulated in at least one of the G-protein subunit null mutants72 . . .
  73. Gao, S. & Nuss, D. L. Distinct roles for two G protein subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption. Proc. Natl Acad. Sci. USA 93, 14122-14127 , (1996) .
    • . . . Disruption of the gene that encodes CPG-1 resulted in a loss of virulence, reduced accumulation of CPGB-1 and a set of phenotypic changes that are similar to, but more severe than, those caused by hypovirus infection72, 73 . . .
  74. Nuss, D. L. Using hypoviruses to probe and perturb signal transduction processing underlying fungal pathogenesis. Plant Cell 8, 1845-1853 , (1996) .
    • . . . These combined results suggested a model in which hypovirus-mediated alterations of cellular signal transduction pathways impede penetration and canker expansion by compromising the ability of the invading fungus to respond appropriately to molecular and environmental cues during the infection process74, 75 . . .
  75. Dawe, A. L. & Nuss, D. L. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu. Rev. Genet. 35, 1-29 , (2001) .
    • . . . Modified with permission from Ref. 75 © (2001) Annual Reviews. . . .
    • . . . Reproduced with permission from Ref. 75 © (2001) Annual Reviews. b | Chimeric virus R12 contains the region of ORF B that extends from the N-terminus of p48 (nucleotide (nt) 2,363) to the Nar I site at nt 5,310 from the severe hypovirus CHV1-EP713 and the remainder of ORF B from the mild hypovirus CHV1-Euro7 . . .
    • . . . These combined results suggested a model in which hypovirus-mediated alterations of cellular signal transduction pathways impede penetration and canker expansion by compromising the ability of the invading fungus to respond appropriately to molecular and environmental cues during the infection process74, 75 . . .
    • . . . The modulation of transcripts for approximately 13.4% of 2,200 different cDNAs was shown following CHV1-EP713 infection, which increased the number of identified hypovirus-responsive host genes from less than 20 (Refs 75, 78) to nearly 300 . . .
    • . . . Contributing factors include barriers to cytoplasmic hypovirus transmission owing to an abundance of vegetative compatibility (vc) types in North America, and the phenotypic characteristics of the hypoviruses that have been used for most attempts at biocontrol (reviewed in Refs 75, 94, 96, 98). . . .
  76. Allen, T. D., Dawe, A. L. & Nuss, D. L. Use of cDNA microarrays to monitor transcriptional responses of the chestnut blight fungus Cryphonectria parasitica to infection by virulence-attenuating hypoviruses. Eukaryotic Cell 2, 1253-1265 , (2003) .
    • . . . The recent development of a C. parasitica cDNA MICROARRAY76 and the availability of several well-characterized C. parasitica signalling mutants are providing new opportunities for studying how hypovirus infection affects cellular signalling. . . .
  77. Soanes, D. M., Skinner, W., Keon, J., Hargraves, J. & Talbot, N. J. Genomics of phytopathogenic fungi and the development of bioinformatics resources. Mol. Plant Microbe Interact. 15, 421-427 , (2002) .
    • . . . As recently as 2002, only 40 C. parasitica genes were deposited in the NCBI (National Center for Biotechnology Information) database, but the construction of an ordered EXPRESSED SEQUENCE TAG (EST) library representing approximately 2,200 C. parasitica genes, that is accessible on the COGEME web site maintained by Exeter University77 (see Online links box), has been a boon to hypovirus research66 . . .
  78. Kang, H. -S. et al. Ordered differential display from Cryphonectria parasitica. Plant Pathol. J. 16, 142-146 , (2000) .
    • . . . The modulation of transcripts for approximately 13.4% of 2,200 different cDNAs was shown following CHV1-EP713 infection, which increased the number of identified hypovirus-responsive host genes from less than 20 (Refs 75, 78) to nearly 300 . . .
  79. Kasahara, S., Wang, P. & Nuss, D. L. Identification of bdm-1, a gene involved in G-protein -subunit function and -subunit accumulation. Proc. Natl Acad. Sci. USA 97, 412-17 , (2000) .
    • . . . This is consistent with reports that deletion of the gene that encodes one of the heterotrimeric G-protein subunits results in the posttranscriptional reduction of the accumulation of a cognate subunit79, 80, 81 . . .
  80. Yang, Q., Poole, S. I. & Borkovich, K. A. A G-protein subunit required for sexual and vegetative development and maintenance of normal G protein levels in Neurospora crassa. Eukaryotic Cell 1, 378-390 , (2002) .
    • . . . This is consistent with reports that deletion of the gene that encodes one of the heterotrimeric G-protein subunits results in the posttranscriptional reduction of the accumulation of a cognate subunit79, 80, 81 . . .
  81. Parsley, T. B., Segers, G. C., Nuss, D. L. & Dawe, A. L. Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third G homologue. Curr. Genet. 43, 24-33 , (2003) .
    • . . . This is consistent with reports that deletion of the gene that encodes one of the heterotrimeric G-protein subunits results in the posttranscriptional reduction of the accumulation of a cognate subunit79, 80, 81 . . .
  82. Monteiro-Vitorello, C. B., Bell, J. A., Fullbright, D. W. & Bertrand, H. A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica. Proc. Natl Acad. Sci. USA 92, 5935-5939 , (1995) .
    • . . . Mitochondrial hypovirulence is a cytoplasmically transmissible form of C. parasitica hypovirulence that is associated with mitochondrial defects82 . . .
    • . . . This isolate and other dsRNA-virus-free hypovirulent C. parasitica strains that were collected from the same area were shown to have increased alternative oxidase activity82, 84, which is a hallmark of mitochondrial dysfunction that is often associated with fungal senescence85 . . .
    • . . . Monteiro-Vitorello and colleagues82 showed that in vitro mutation of mitochondrial DNA (mtDNA) in C. parasitica strain EP155 induced transmissible hypovirulence . . .
  83. Fulbright, D. W. A cytoplasmic hypovirulent strain of Endothia parasitica without double-stranded RNA. Phytopathology 75, 1328 , (1985) .
    • . . . Fulbright reported the first isolation of a dsRNA-virus-free hypovirulent strain of C. parasitica from a healing canker in the Kellogg Forest in Michigan83 . . .
  84. Mahanti, N., Bertrand, H., Monteiro-Vitorello, C. & Fulbright, D. W. Elevated mitochondrial alternative oxidase activity in dsRNA-free, hypovirulent isolates of Cryphonectria parasitica. Physiol. Mol. Plant Pathol. 42, 455-463 , (1993) .
    • . . . This isolate and other dsRNA-virus-free hypovirulent C. parasitica strains that were collected from the same area were shown to have increased alternative oxidase activity82, 84, which is a hallmark of mitochondrial dysfunction that is often associated with fungal senescence85 . . .
  85. Griffiths, A. J. F. Mitochondrial inheritance in filamentous fungi. J. Genet. 75, 403-414 , (1996) .
    • . . . This isolate and other dsRNA-virus-free hypovirulent C. parasitica strains that were collected from the same area were shown to have increased alternative oxidase activity82, 84, which is a hallmark of mitochondrial dysfunction that is often associated with fungal senescence85 . . .
  86. Allen, T. D. & Nuss, D. L. Linkage between mitochondrial hypovirulence and viral hypovirulence in the chestnut blight fungus revealed by cDNA microarray analysis. Eukaryotic Cell 3, 1227-1232 , (2004) .
    • . . . Approximately 10% of the 2,220 host genes studied using transcriptional profiling were modulated by the mit-2 mtDNA mutation86 . . .
  87. Burtrand, H. Role of mitochondrial DNA in the senescence and hypovirulence of fungi and potential for plant disease control. Annu. Rev. Phytopathol. 38, 397-422.Excellent review of the relationship between mitochondrial dysfunction and the attenuation of fungal virulence known as mitochondrial hypovirulence , (2000) .
    • . . . However, there is no evidence that mitochondrial dysfunction is a common characteristic of hypovirus-infected C. parasitica strains87 . . .
  88. Dementhon, K. et al. Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina. Eukaryotic Cell 2, 238-246 , (2003) .
    • . . . A possible candidate is the highly conserved TOR pathway that integrates cellular responses to starvation, mitochondrial dysfunction, changes in ATP levels and other stress conditions88, 89, 90 . . .
  89. Dennis, P. et al. Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102-1105 , (2001) .
    • . . . A possible candidate is the highly conserved TOR pathway that integrates cellular responses to starvation, mitochondrial dysfunction, changes in ATP levels and other stress conditions88, 89, 90 . . .
  90. Desai, B., Myers, B. & Schreiber, S. FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc. Natl Acad. Sci. USA 99, 4319-4324 , (2002) .
    • . . . A possible candidate is the highly conserved TOR pathway that integrates cellular responses to starvation, mitochondrial dysfunction, changes in ATP levels and other stress conditions88, 89, 90 . . .
  91. Biraghi, A. Possible active resistance to Endothia parasitica in Castanea sativa in Rep. Congr. Int. Union For. Res. Org. 11th. 149-157 (International Union of Forest Research Organization, Rome, 1953) , .
    • . . . The French Ministry of Agriculture employed hypovirulent C. parasitica strains, first identified in Italy in the 1950's (Ref. 91), in a large biocontrol programme during the 1960's and 1970's (Ref. 92) — long before Day and co-workers reported the isolation of viral-like dsRNAs from a hypovirulent C. parasitica isolate93 . . .
  92. Grente, J. & Berthelay-Sauret, S. in Proceedings of the American Chestnut Symposium, (eds MacDonald, W. L., Cech, F. C., Luchok, J. & Smith, C.) 30-34 (West Virginia University, Morgantown, 1978) , .
    • . . . The French Ministry of Agriculture employed hypovirulent C. parasitica strains, first identified in Italy in the 1950's (Ref. 91), in a large biocontrol programme during the 1960's and 1970's (Ref. 92) — long before Day and co-workers reported the isolation of viral-like dsRNAs from a hypovirulent C. parasitica isolate93 . . .
  93. Day, P. R., Dodds, J. A., Elliston, J. E., Jaynes, R. A., & Anagnostakis, S. L. Double-stranded RNA in Endothia parasitica. Phytopathology 68, 1391-1396 , (1977) .
    • . . . The French Ministry of Agriculture employed hypovirulent C. parasitica strains, first identified in Italy in the 1950's (Ref. 91), in a large biocontrol programme during the 1960's and 1970's (Ref. 92) — long before Day and co-workers reported the isolation of viral-like dsRNAs from a hypovirulent C. parasitica isolate93 . . .
  94. Heiniger, U., & Rigling, D. Biological control of chestnut blight in Europe. Annu. Rev. Phytopathol. 32, 581-599.Excellent review of the history and current picture of chestnut blight and hypovirulence in Europe , (1994) .
    • . . . Hypovirulence continues to be used for the control of chestnut blight in European chestnut orchards and has been credited with reducing the severity and extent of the chestnut blight epidemic as a result of natural spread through European chestnut coppice forests (see Ref. 94 for an extensive review) . . .
    • . . . Contributing factors include barriers to cytoplasmic hypovirus transmission owing to an abundance of vegetative compatibility (vc) types in North America, and the phenotypic characteristics of the hypoviruses that have been used for most attempts at biocontrol (reviewed in Refs 75, 94, 96, 98). . . .
  95. Griffin, G. J. Chestnut blight and its control. Hortic. Rev. 8, 291-336 , (1986) .
    • . . . Efforts to establish effective biocontrol in North American forest ecosystems by the artificial introduction of hypovirulent C. parasitica strains have so far been unsuccessful2, 95, 96 . . .
  96. MacDonald, W. L. & Fulbright, D. W. Biological control of chestnut blight: use and limitation of transmissible hypovirulence. Plant Dis. 75, 656-661.This review provides an excellent perspective on the importance of balancing the effects of hypovirus infection on ecological fitness and virulence attenuation for successful hypovirulence-mediated biological control , (1991) .
    • . . . Efforts to establish effective biocontrol in North American forest ecosystems by the artificial introduction of hypovirulent C. parasitica strains have so far been unsuccessful2, 95, 96 . . .
    • . . . Naturally occurring hypovirulence does, however, contribute to chestnut tree survival in the state of Michigan, a region outside the natural range of the American Chestnut96, 97 . . .
    • . . . Contributing factors include barriers to cytoplasmic hypovirus transmission owing to an abundance of vegetative compatibility (vc) types in North America, and the phenotypic characteristics of the hypoviruses that have been used for most attempts at biocontrol (reviewed in Refs 75, 94, 96, 98). . . .
    • . . . As noted by MacDonald and Fulbright96, CHV1-EP713, similar to most hypoviruses that have been used for biocontrol efforts in North America, is predicted to perform poorly as a biocontrol agent because it severely reduces the ability of the fungal host to colonize, expand and produce asexual spores on chestnut tissue . . .
  97. Fulbright, D. W., Weidlich, W. H., Haufler, Z., Thomas, C. S. & Paul, C. P. Chestnut blight and recovering American chestnut trees in Michigan. Can. J. Bot. 61, 3144-3171 , (1983) .
    • . . . Naturally occurring hypovirulence does, however, contribute to chestnut tree survival in the state of Michigan, a region outside the natural range of the American Chestnut96, 97 . . .
  98. Milgroom, M. G. & Cortesi, P. Biological control of chestnut blight with hypovirulence: a critical analysis. Annu. Rev. Phytopathol. 42, 311-338.Recent critical assessment of the effectiveness of hypovirulence in the management of chestnut blight , (2004) .
    • . . . A recent review by Milgroom and Cortesi98 critically assessed the effectiveness of hypovirulence in the management of chestnut blight. . . .
    • . . . Contributing factors include barriers to cytoplasmic hypovirus transmission owing to an abundance of vegetative compatibility (vc) types in North America, and the phenotypic characteristics of the hypoviruses that have been used for most attempts at biocontrol (reviewed in Refs 75, 94, 96, 98). . . .
    • . . . Although vegetative incompatibility presents barriers to hypovirus transmission, several recent examples of discrepancies in results observed for virus transmission in the laboratory and the field98 indicate that vegetative incompatibility might be less important in inhibiting virus transmission in natural C. parasitica populations than predicted by laboratory studies . . .
    • . . . Factors that reduce the rate of expansion of a chestnut blight epidemic, such as the lower blight susceptibility of the European chestnut, increase the probability of hypovirus transmission through the fungal population98, 111 . . .
  99. Cortesi, P. & Milgroom, M. G. Genetics of vegetative incompatibility in Cryphonectria parasitica. Appl. Environ. Microbiol. 64, 2988-2994 , (1998) .
    • . . . The VEGETATIVE INCOMPATIBILITY system in C. parasitica is controlled by at least six vic loci, each consisting of two alleles99 . . .
    • . . . Fungal strains with identical alleles at all (or most) of the vic loci99 readily anastomose, allowing virus transmission (Fig. 1) . . .
  100. Liu, Y. -C. & Milgroom, M. G. Correlation between hypovirus transmission and the number of vegetative incompatibility (vic) genes different among isolates from natural populations of Cryphonectria parasitica. Phytopathology 86, 79-86 , (1996) .
    • . . . Therefore, an introduced hypovirus is predicted to spread more slowly through a C. parasitica population with a high level of vc diversity100, 101, as is found in most North American forest ecosystems, than through a population of low diversity, as is usually found in Europe100, 102, 103 . . .
  101. Cortesi, P., McCulloch, C. E., Song, H., Lin, H. & Milgroom, M. G. Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica. Genetics 159, 107-118 , (2001) .
    • . . . Therefore, an introduced hypovirus is predicted to spread more slowly through a C. parasitica population with a high level of vc diversity100, 101, as is found in most North American forest ecosystems, than through a population of low diversity, as is usually found in Europe100, 102, 103 . . .
  102. Anagnostakis, S. L. & Kranz, J. Population dynamics of Cryphonectria parasitica in a mixed-hardwood forest in Connecticut. Phytopathology 77, 751-754 , (1987) .
    • . . . Therefore, an introduced hypovirus is predicted to spread more slowly through a C. parasitica population with a high level of vc diversity100, 101, as is found in most North American forest ecosystems, than through a population of low diversity, as is usually found in Europe100, 102, 103 . . .
  103. Bissegger, M., Rigling, D. & Heiniger, U. Population structure and disease development of Cryphonectria parasitica in European chestnut forests in the presence of natural hypovirulence. Phytopathology 87, 51-59 , (1997) .
    • . . . Therefore, an introduced hypovirus is predicted to spread more slowly through a C. parasitica population with a high level of vc diversity100, 101, as is found in most North American forest ecosystems, than through a population of low diversity, as is usually found in Europe100, 102, 103 . . .
  104. Chen, B., Chen, C. -H., Bowman, B. H. & Nuss, D. L. Phenotypic changes associated with wild-type and mutant hypovirus RNA transfection of plant pathogenic fungi phylogenetically related to Cryphonectria parasitica. Phytopathology 86, 301-310 , (1996) .
    • . . . Hypovirus spread is also influenced by the efficiency of transmission through conidia (Fig. 1), which can vary from 1% to 99% depending on the hypovirus–fungal host combination43, 104, 105 . . .
  105. Shain, L. & Miller, J. B. Movement of cytoplasmic hypovirulence agents in chestnut blight cankers. Can. J. Bot. 70, 557-561 , (1992) .
    • . . . Hypovirus spread is also influenced by the efficiency of transmission through conidia (Fig. 1), which can vary from 1% to 99% depending on the hypovirus–fungal host combination43, 104, 105 . . .
  106. Chen, B., Choi, G. H. & Nuss, D. L. Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus. EMBO J. 12, 2991-2998 , (1993) .
    • . . . By contrast, the hypovirus cDNA copy that is present in the nucleus of transgenic hypovirulent strains is inherited by a portion of the ascospore progeny, followed by production of cDNA-derived cytoplasmically replicating viral RNA106 . . .
  107. Liu, Y. -C., Durrett, R. & Milgroom, M. G. A spatially-structured stochastic model to simulate heterogeneous transmission of viruses in fungal populations. Ecol. Modell. 127, 291-308 , (2000) .
    • . . . A mathematical model107 has predicted that transgenic strains provide only a small improvement over non-transgenic strains in viral invasion . . .
  108. Anagnostakis, S. L., Chen, B., Geletka, L. M. & Nuss, D. L. Hypovirus transmission to ascospore progeny by field-released transgenic hypovirulent strains of Cryphonectria parasitica. Phytopathology 88, 598-604 , (1998) .
    • . . . A single-season release in the Housatonic State Forest in Connecticut in 1994 (Ref. 108) clearly established that a transgenic hypovirulent C. parasitica strain can transmit hypovirus to ascospore progeny through an intermediate cDNA copy under field conditions and showed that cDNA-derived hypovirus RNA is cytoplasmically transmitted to indigenous, virulent C. parasitica strains independent of the integrated viral cDNA copy . . .
  109. Root, C., et al. Multiseasonal field release and spermatization of transgenic hypovirulent strains of Cryphonectria parasitica containing cDNA copies of the highly debilitating hypovirus CHV1-EP713. For. Pathol. (in the press) , .
    • . . . Results of two subsequent multiseasonal release trials109 confirmed the earlier reports and illustrated the need for the development of improved formulation and application methods . . .
    • . . . For example, the application of transgenic conidia as a water suspension from a backpack sprayer was inefficient in establishing either fungal colonization of chestnut tissue or spermatization of cankers to produce transgenic ascospores109 . . .
  110. Gobbin, D., Hoegger, P. J., Heiniger, U. & Rigling, D. Sequence variation and evolution of Cryphonectria hypovirus 1 (CHV1) in Europe. Virus Res. 97, 39-46 , (2003) .
    • . . . Consistent with this prediction, a recent survey of hypoviruses in regions of Europe with a high incidence of hypovirulence found little evidence of severe CHV1-EP713-related hypoviruses and a prevalence of mild CHV1-Euro7-related hypoviruses41, 110 . . .
  111. Graves, A. H. Relative blight resistance in species and hybrids of Castanea. Phytopathology 40, 1125-1131 , (1950) .
    • . . . Factors that reduce the rate of expansion of a chestnut blight epidemic, such as the lower blight susceptibility of the European chestnut, increase the probability of hypovirus transmission through the fungal population98, 111 . . .
  112. Hebard, F. V. Inheritance of juvenile leaf and stem morphological traits in crosses of Chinese and American chestnut. J. Hered. 85, 440-446 , (1994) .
    • . . . In this regard, the use of hypovirulence to manage chestnut blight in North America might be most effective as part of an integrated approach with the ongoing chestnut-blight-resistance breeding programme112 or with efforts to produce blight-resistant chestnut using transgenic technology113. . . .
  113. Connors, B. J., Laun, N. P., Maynard, C. A. & Powell, W. A. Molecular characterization of a gene encoding a cystatin expressed in the stems of American chestnut (Castanea dentate). Planta 3, 510-514 , (2002) .
    • . . . In this regard, the use of hypovirulence to manage chestnut blight in North America might be most effective as part of an integrated approach with the ongoing chestnut-blight-resistance breeding programme112 or with efforts to produce blight-resistant chestnut using transgenic technology113. . . .
  114. Jian, J. H., Lakshman, D. K. & Tavantzis, S. M. Association of distinct double-stranded RNAs with enhanced or diminished virulence in Rhizoctonia solani infecting potato. Mol. Plant Microbe Interact. 10, 1002-1009 , (1997) .
  115. Jian, J. H., Lakshman, D. K. & Tavantzis, S. M. A virulence-associated, 6.4-kb, double-stranded RNA from Rhizoctonia solani is phylogenetically related to plant bromoviruses and electron transport enzymes. Mol. Plant Microbe Interact. 11, 601-609 , (1998) .
  116. Liu, C., Lakshman, D. K. & Tavantzis, S. M. Quinic acid induces hypovirulence and expression of a hypovirulence-associated double-stranded RNA in Rhizoctonia solani. Curr. Genet. 43, 103-111 , (2003) .
  117. Polashock, J. J., Bedker, P. J. & Hillman, B. I. Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination. Mol. Gen. Genet. 256, 566-571 , (1997) .
    • . . . However, the Cryphonectria parasitica mitovirus NB631 has been shown to be efficiently transmitted to sexual spores (ascospores)117. dsRNA, double-stranded RNA; ssRNA, single-stranded RNA. . . .
  118. Shapira, R. & Nuss, D. L. Gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities. J. Biol. Chem. 266, 19419-19425 , (1991) .
    • . . . Cleavage, in this case, occurs between Gly418 and Ala419 of the target sequence LVG AEE (Ref. 118) . . .
  119. Nuss, D. L., Chen, B., Geletka, L. M., Parsley, T. B. & Suzuki, N. in Molecular Biology of double-stranded RNA: Concepts and Applications in Agriculture, Forestry and Medicine (ed. Tavantzis, S.) 145-163 (CRC, Boca Raton, 2002) , .
    • . . . Modified with permission from Ref. 119 © (2002) CRC. . . .
  120. Buck, K. W. & Brasir, C. M. in Molecular Biology of double-stranded RNA: Concepts and Applications in Agriculture, Forestry and Medicine (ed. Tavantzis, S.) 165-190 (CRC, Boca Raton, 2002) , .
  121. Jiang, D. & Ghabrial, S. A. Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus. J. Gen. Virol. 85, 2111-2121 , (2004) .
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